Embodiments of the present invention relate to microscopes, and more particularly to a digital optical microscope that autofocuses on a sample.
Digital optical microscopes are used to observe a wide variety of samples. Recently, a need has emerged to digitally record images of biological samples such as biopsy tissue on histopathologic slides for archiving, telepathology and rapid information retrieval. Although the samples are relatively thin, on the order of 5 microns, high power objective lenses with a numerical aperture greater than 0.5 typically have a depth of field that is even smaller. Furthermore, the sample, coverslip and slide may have variable thickness, the sample may grow or move, and the microscope may exhibit mechanical instability, scanning stage misalignment and thermal expansion.
Therefore, in order to keep the sample in focus (along the optical axis in the Z direction) as the microscope scans the sample and relocates the field of view to take snapshots of the sample at different lateral locations (in the XY focal plane), the microscope needs to autofocus on the sample to keep the objective lens within a suitable focal distance of the sample to generate high quality images.
Rapid autofocusing is important in automated biological and biomedical applications such as high-throughput pharmaceutical screening and large-scale autonomous microrobotic cell manipulation. Rapid autofocusing is also important in other applications such as integrated circuit chip inspection and microassembly of hybrid microelectromechanical systems (MEMS). Thus, rapid autofocusing is highly desirable in real-time image acquisition applications that cannot afford considerable time delays to adjust the focal distance between snapshots of the sample.
Conventional microscopes perform autofocusing by directing a laser beam at the sample, measuring a reflection of the laser beam off the sample to provide a single reference point, and using a feedback loop to adjust the focal distance. Although this approach provides rapid autofocusing, the single reference point may lack sufficient information for accurate autofocusing.
Conventional microscopes also perform autofocusing by obtaining multiple images at multiple focal distances, determining a quantitative characteristic for each image, determining an optimal focal distance based on the quantitative characteristic and using a feedback loop to adjust the focal distance. Although this approach provides accurate autofocusing, acquiring the multiple images may create time delays that prevent rapid autofocusing.
Therefore, there is a need for a microscope that performs accurate rapid autofocusing.